If it's any use, I wrote an article on how to hack the other partner in yoghurt fermentation, S.thermophilus:
http://www.indiebiotech.com/?p=152
"Meredith L. Patterson" <clonearmy@gmail.com> wrote:
>"Thermal" means "heat", but the important bit for this one is step 4,
>electroporation. If you've got access to a Bio-Rad GenePulser then
>that's awesome; there've been some discussions of DIY electroporation
>using piezo lighters on this mailing list, but I don't know if anyone
>has successfully transformed bacteria with one yet. (Simon? Nathan? I
>seem to recall y'all discussing this. Also I've owed John Griessen an
>email about this for months now, but real life has intervened more
>than it typically does.)
>
>Lactobacilli aren't terribly amenable to heat shock. There are some
>protocols in the patent literature involving PEG but they're
>apparently quite touchy.
>
>Cheers,
>--mlp
>
>On Tue, Mar 6, 2012 at 8:01 PM, Petfixer71 <afrishman71@hotmail.com>
>wrote:
>> |One of the first "projects" I want to try on my own (after doing
>some
>> basic "Kit" transformations) is "hacking yogurt".
>>
>> I have a couple question regarding the below protocol:
>>
>> 1) Thermal Shock- is suggested for this transformation.....Can I do a
>> "heat shock" instead? with same results?
>>
>> 2) Plasmids- pJK650 and pLEM415....can I purchase these plasmids
>> online? I need a resource for ordering plasmids?
>>
>> 3) Can the plasmids be custom ordered? with DNA of my choice?
>>
>> I would love some feedback.
>>
>> Thanks,
>>
>> Andrew
>>
>> Below is the protocol:
>> _______________________________________________________________
>>
>> Materials
>>
>> List reagents, supplies and equipment necessary to perform the
>> protocol here. For those materials which have their own OWW pages,
>> link to that page. Alternatively, links to the suppliers' page on
>that
>> material are also appropriate.
>>
>> * MRS media
>> * Electroporation Buffer:(0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4;
>> PH 6)
>> * Milk Medium: (0.2 M sucrose, 5% skim milk, 0.1% yeast extract,
>> 1% Casamino Acids, 25mM MgCl2)
>> * Erythromycin, Chloramphenicol, other antibiotics may be needed
>> for selection
>> * Gene Pulser and Pulse Controller Electrophoration apparatus
>> * Gaspak or method to generate anaerobic conditions
>> * Strain of compatible Lactobacillus - For list of compatible
>> strains/plasmids, please click [here]
>>
>> Procedure
>>
>> Estimated time for procedure: 3-4 days
>>
>> 1. CELL CULTURE
>> 1. Inoculate serial dilutions of fresh bacterial culture into
>> 100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.
>> 2. Harvest 10 ml of culture cells at beginning of stationary
>> phase (optical density at 600 nm, 1.7) by centrifugation (need ~2.3mL
>> of culture per electroporation)
>> 2. WASH BUFFER
>> 1. `Wash bacteria once with 100 ml of cold electroporation
>> buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)
>> 2. Wash bacteria twice with 30 ml of cold EB
>> 3. THERMAL SHOCK
>> 1. Resuspend cells in EB to an optical density at 600 nm of
>> about 50
>> 2. Incubate cell suspension at 45°C for 20 min then keep on
>> ice for 10 min
>> 4. ELECTRICAL PULSE
>> 1. Mix 80 ?l of cell suspension with 0.3 to 2 ?g of plasmid
>> DNA
>> 2. Subject sample to a 1-kV, 800-Ω, 25-?F electric pulse in a
>> 0.2-cm cuvette by using a Gene Pulser and a Pulse Controller
>> apparatus.
>> 5. EXPRESSION
>> 1. Immediately add 2 milliliters of milk medium (0.2 M
>> sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM
>> MgCl2)
>>
>> *Derek Ju 04:28, 29 October 2008 (EDT): I have found that adding MRS
>> works just as well (and saves the step of making the milk medium)
>>
>> 1. PLATING, SELECTION
>> 1. Incubate cells for 3h at 37°C before plating on MRS agar
>> supplemented with antibiotics. Add antibiotic erythromycin at
>> concentration 7.5 ?g/ml and chloramphenicol at concentration 7.5
>?g/ml
>> depending on plasmid resistance gene
>> 2. Incubate plates at 37°C for 2 to 3 days under anaerobic
>> conditions in jars containing GasPak
>>
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